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研究生: 邱勝暉
Sheng-Hui Chiu
論文名稱: 微波輔助異原子摻雜水相碳量子點之製備及其生醫應用
Rapid microwave-assisted synthesis of heteroatom-doped carbon quantum dot for tumor-targeted bioimaging and anticancer drug delivery applications
指導教授: 張家耀
Jia-Yaw Chang
口試委員: 何郡軒
Jinn-Hsuan Ho
黃志清
Chih-Ching Huang
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2016
畢業學年度: 104
語文別: 中文
論文頁數: 165
中文關鍵詞: 碳量子點異原子摻雜生物顯影磁振顯影
外文關鍵詞: carbon quantum dot, heteroatom doping, bioimaging, magnetic resonance imaging
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本研究以微波輔助水熱法合成低毒性之碳量子點(Carbon quantum dot,CQD),藉由異原子摻雜提升碳量子點之螢光強度以及賦予碳量子點新穎特性。
第一部分:選擇硫(Sulphur,S)、硼(Boron,B)與磷(Phosphorous,P)作為摻雜至碳量子點結構之異原子,提升其螢光量子產率,並藉由不同的實驗參數以得到合成之最適化條件,僅需10 min反應時間即可得到螢光量子產率高達55%之P摻雜碳量子點(P-CQD),且具有良好的螢光穩定性。接著以玻尿酸(Hualuronic acid,HA)作為功能性分子,藉由EDC/sulfo-NHS化學系統將玻尿酸與P-CQD結合形成P-CQD-HA,透過與小鼠黑色素瘤細胞(B16F1)表面玻尿酸受體之專一性作用,P-CQD-HA可經由B16F1表面玻尿酸受體介導之胞飲作用而進入細胞內,達到螢光標靶顯影之效果。最後藉由斑馬魚活體實驗證實P-CQD-HA應用於生物體內螢光顯影之可行性。
第二部分: 選擇釓(Gadolinium,Gd)作為摻雜至碳量子點結構之異原子,Gd為順磁性材料,可作為磁振顯影之正向T1顯影劑而使顯影圖更加明亮。藉由微波輔助加熱之水熱法,僅需10 min反應時間即可得Gd異原子摻雜之磁性碳量子點(GdCQD),相較於市售Gd-DTPA,本研究製備之GdCQD具有更明顯的影像對比效果,於T1之遲緩速率值(r1)為9.02 mM-1s-1,搭配碳量子點本身具有的優異螢光特性,GdCQD可同時提供螢光顯影與磁振顯影而達到雙重顯影功能。接著藉由EDC/sulfo-NHS化學系統使GdCQD共價鍵結葉酸分子(Folic acid,FA)而形成GdCQD-FA,研究結果顯示GdCQD-FA具有低生物毒性,且可透過子宮頸癌細胞(Hela)表面葉酸受體介導之胞飲作用而進入細胞內,同時提供癌細胞標靶、螢光顯影與磁振顯影之功能,於斑馬魚活體實驗之結果也證實GdCQD-FA應用於生物體內螢光顯影之可行性。最後,藉由GdCQD-FA與抗癌藥物Doxorubicin (DOX)間之-作用力將DOX附載於碳量子點上(GdCQD-FA/DOX),其附載量可達75%,GdCQD-FA/DOX可透過Hela表面葉酸受體介導之胞飲作用而進入細胞內,並藉由癌細胞內pH值變化驅動DOX釋放,使其能夠作為藥物載體並達到藥物治療之效果。


Carbon quantum dot (CQD) exhibits unique properties such as tunable fluorescence, high photostability, chemical inertness, low toxicity, and biocompatibility, which are beneficial for bioimaging application. Herein, we synthesized heteroatom doped CQD by using a one-pot microwave method. Doping heteroatom elements into CQD could modulate the fluorescent properties and improve the functionality.
Part Ⅰ
Sulphur (S), boron (B), and phosphorous (P) heteroatom doped CQD (S-CQD, B-CQD, and P-CQD) were synthesized within 10 min by using a one-pot microwave method. Doping S, B, and P heteroatom into CQD could increase the quantum yield from 5.9% to 22.5, 24.9, and 51.7%, respectively. P-CQD was conjugated with hyaluronic acid forming P-CQD-HA for targeting fluorescence imaging. The in vitro and in vivo studies confirmed the high biocompatibility and low toxicity of P-CQD-HA. The targeting capabilities of P-CQD-HA were confirmed in B16F1 and HeLa cells using in vitro fluorescence imaging. Additionally, we successfully demonstrated in vivo fluorescence imaging of the P-CQD-HA, using zebrafish as an animal model.
Part Ⅱ
Gadolinium (Gd) heteroatom doped CQD (GdCQD) was synthesized within 10 min by using a one-pot microwave method. Our results showed that the synthesized multifunctional GdCQD has excellent structural, fluorescent, and magnetic properties. GdCQD enhanced the MR response as compared to that for commercial Gd-DTPA. The multifunctional GdCQD was conjugated with folic acid forming GdCQD-FA for targeting dual modal fluorescence/magnetic resonance (MR) imaging. The in vitro and in vivo studies confirmed the high biocompatibility and low toxicity of GdCQD-FA. The targeting capabilities of GdCQD-FA were confirmed in HeLa and HePG2 cells using in vitro fluorescence and MR dual modality imaging. We successfully demonstrated in vivo fluorescence imaging of the GdCQD-FA, using zebrafish as an animal model. Additionally, an anticancer drug, doxorubicin (DOX), was incorporated into the nanocomposite forming GdCQD-FA/DOX, which enables targeted drug delivery. Importantly, the prepared multifunctional GdCQD-FA/DOX showed a high quantity of DOX loading capacity (about 75%) and pH-sensitive drug release. The uptake into cancer cells and the intracellular location of the GdCQD-FA/DOX were observed by confocal laser scanning microscopy. Therefore, the GdCQD-FA/DOX nanocomposite is a promising strategy for potential application as a dual modal diagnosis agent and drug delivery system for chemotherapy.

摘要 Abstract 致謝 總目錄 表目錄 圖目錄 第一章、序論 1.1 前言 1.2 研究動機與目的 第二章、理論基礎與文獻回顧 2.1 量子點(Quantum Dot,QD) 2.1.1 量子點之介紹 2.1.2 量子點於生醫應用上之發展與限制 2.2 碳量子點(Carbon Quantum dot,CQD) 2.2.1 碳量子點之起源 2.2.2 碳量子點之合成方式 2.2.3 碳量子點之光學性質 2.2.4 碳量子點之發光機制 2.2.5 異原子摻雜之碳量子點 2.2.6 碳量子點之生物顯影應用 2.2.7 碳量子點之表面功能化與特異性標靶顯影 2.2.8 雙重顯影功能之碳量子點 2.2.9 碳量子點作為藥物載體與藥物治療之應用 第三章、實驗儀器與方法 3.1 實驗藥品 3.2 實驗儀器 3.3 實驗步驟 3.3.1 碳量子點之合成(Synthesis of CQD) 3.3.2 異原子摻雜碳量子點之合成(Synthesis of heteroatom doped CQD) 3.3.3 磁性碳量子點之合成(Synthesis of GdCQD) 3.3.4 以葉酸功能化磁性碳量子點之合成(Synthesis of GdCQD-FA) 3.3.5 以GdCQD-FA作為藥物載體附載抗癌藥物Doxorubicin (Synthesis of GdCQD-FA/DOX) 3.3.6 以玻尿酸功能化磷摻雜碳量子點之合成(Synthesis of PCQD-HA) 3.4 細胞培養與細胞實驗 3.4.1 培養液(Medium)與PBS之配製 3.4.2 解凍細胞(Cell Defrost) 3.4.3 繼代培養(Cell Culture) 3.4.4 細胞計數(Cell Counting) 3.4.5 冷凍細胞(Cell Cryopreservation) 3.4.6 碳量子點於細胞之螢光顯影試片製作 3.4.7 碳量子點於細胞之磁振顯影試片製作 3.4.8 碳量子點於細胞之體外毒性測試 3.4.9 碳量子點於斑馬魚之體內毒性測試 3.4.10 碳量子點於斑馬魚之螢光顯影與代謝 第四章、結果與討論 4.1硫、硼與磷異原子摻雜之碳量子點(S-CQD、B-CQD、P-CQD) 4.1.1 S-CQD、B-CQD、P-CQD之實驗介紹 4.1.2 S-CQD之最適化合成條件 4.1.3 B-CQD與P-CQD之最適化合成條件 4.1.4 S-CQD、B-CQD與P-CQD之材料鑑定分析 4.1.5 P-CQD之表面功能化與鑑定分析 4.1.6 P-CQD-HA對於細胞之體外毒性測試與斑馬魚之體內毒性測試 4.1.7 P-CQD-HA之生物顯影應用 4.2 釓異原子摻雜之磁性碳量子點(GdCQD) 4.2.1 GdCQD之實驗介紹 4.2.2 GdCQD之最適化合成條件 4.2.3 GdCQD之材料鑑定分析 4.2.4 GdCQD之表面功能化與鑑定分析 4.2.5 GdCQD-FA對於細胞之體外毒性測試與斑馬魚之體內毒性測試 4.2.6 GdCQD-FA之生物顯影應用 4.2.7 GdCQD-FA附載抗癌藥物DOX之鑑定分析與生物應用 第五章、結論與未來展望 第六章、參考文獻

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